ANTICANCER RESEARCH 36 : 5827-5834 (2016) doi:10.21873/anticanres.11167

Isoflavone Extracts Enhance the Effect of Epidermal Growth Factor Receptor Inhibitors in NSCLC Cell Lines RITA AMBROSIO 1* , MARIA NEVE OMBRA 2* , CESARE GRIDELLI 1, GIANLUCA PICARIELLO 2, MICHELE DI STASIO 2 and MARIA G. VOLPE 2

1Division of Medical Oncology, S.G. Moscati Hospital, Avellino, Italy; 2Institute of Food Sciences, National Research Council, Avellino, Italy

Abstract. Aim: We investigated the effects of the NSCLCs (1-3). Irreversible inhibitors such as afatinib and pharmacological inhibition in vitro of epidermal growth AZD9291 constitute second- and third-generation EGFR factor receptor (EGFR) in combination with isoflavones. inhibitors. Progression-free survival (PFS) of patients with Materials and Methods: Four anticancer drugs (erlotinib, EGFR mutation receiving gefitinib, erlotinib or afatinib is gefitinib, afatinib and AZD9291) were combined with soy improved when compared with standard chemotherapy, as and red clover isoflavone extracts and used in cellular demonstrated with randomized trials (4). Unfavorably, TKI proliferation assays. The antitumor activity of inhibitors therapies may become ineffective due to acquired resistance alone and in combination with isoflavone extracts was that may develop after a duration of response of ~10 months compared on three non-small cell lung cancer (NSCLC) cell up to 1 year (5, 6). A secondary mutation in exon 20 of lines with affiant EGFR genotype: A549 (EGFR wt); H1795 EGFR within the residue at position 790 inducing a (EGFR T790M); HCC827 (EGFR del E746-A750). Results: threonine to methionine substitution (T790M) was Combined treatment with extracts significantly enhanced the determined as the first cause of resistance identified on a antiproliferative activity of all inhibitors against these cell genetic basis. T790M causes resistance through changes in lines. Bioactive compounds of extracts may synergize the adenosine triphosphate (ATP) affinity because T790M antitumor efficacy of the inhibitors. Conclusion: To date, as mutation restores the ATP affinity of the kinase, re- far as we are aware, this is the first report of the combined establishing ATP as the favored substrate rather than the effect of isoflavone extracts and EGFR inhibitors on human administered TKI (7). Second-generation EGFR TKIs, such NCSLC cell growth. Sequential treatment with these drugs afatinib, form irreversible covalent bonds with EGFR and are combined with isoflavones may represent the basis for a new potentially effective in vitro against cells with EGFR therapeutic approach. T790M. Third-generation EGFR TKIs, such as AZD9291/are being developed to overcome resistance to first- and second- Epidermal growth factor receptor (EGFR)-targeted therapies generation EGFR TKIs. In preclinical studies, AZD9291 was using small molecules, such as tyrosine kinase inhibitors active both in vitro and in vivo against cell lines and murine (TKI) have recently been approved for non-small cell lung models harboring the EGFR T790M mutation (8). Very cancer (NSCLC). In particular, two EGFR tyrosine kinase promising results from an ongoing phase I study on inhibitors (TKI), gefitinib (ZD1839, Iressa) and erlotinib AZD9291 have been reported (9). (OSI774, Tarceva), represent the first generation of Recently, natural dietary compounds have captured molecular-targeted agents developed for the treatment of attention because of their synergistic effects with anticancer drugs against various types of cancer. Natural phytochemicals may accomplish their antitumoral effect through targeting diverse cancer cell signaling pathways, *These Authors contributed equally to this study. giving rise to cell-cycle block and apoptosis, or regulating antioxidant status and detoxification (10). Much evidence Correspondence to: Dr Maria Grazia Volpe, Institute of Food has shown that isoflavones exert their pleiotropic effects on Sciences – CNR, Via Roma, 64, 83100 Avellino, AV, Italy. Tel: +39 0825299411, Fax: +39 0825781585, e-mail: [email protected] cancer cells by interfering in cellular signaling pathways including nuclear factor kappa-light-chain-enhancer of Key Words: Isoflavones, non-small cell lung cancer, antiproliferative activated B-cells, serine/threonine kinase, mitogen-activated activity, tyrosine kinase inhibitors. protein kinase, and p53.

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Isoflavones are principally recovered in members of the CA, USA). Total flavonoid content was expressed as mg catechin Leguminosae family (11). Genistein is the most extensively equivalents (CAE, in mg) per gram of dry material. All samples analyzed flavonoid, it is involved in numerous biological were analyzed in triplicates. actions with potential beneficial effects, rightly becoming a Determination of antioxidant activity. The 2,2-diphenyl-1- focus for research (12). Genistein is also a well-known TKI picrylhydrazyl (DPPH) free radical-scavenging activity assay was and induces apoptosis in several types of cancer, such as performed as described by Rostagno et al. (15). Briefly 20 μl of lymphoma, leukemia, breast, prostate, head and neck, extract (1 mg/ml) in absolute ethanol was added to 180 μl of DPPH pancreatic and lung cancer (13). reagent in a 96-well plate. The absorbance was measured at 517 nm In the present study, the effect of gefitinib, erlotinib, after 90 min, with a microplate reader (Biotek). Experiments were afatinib and AZD9291 (selective EGFR TKIs) combined performed in triplicates. The results were expressed as percentage with isoflavones from soy and red clover was assayed on a inhibition (I%), which was calculated using the following formula: panel of NSCLC cell lines by using 3-(4,5-dimethylthiazol- I%=[(A DPPH −A Sample )/A DPPH ]×100 2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet assays. Our aim was to evaluate in vitro whether these where: A DPPH is the absorbance of the control DPPH at time t=0’ isoflavone extracts synergize with TKIs to inhibit NSCLC and cell growth. ASample is the absorbance of the sample after time t=90’.

Materials and Methods High-performance liquid chromatography (HPLC) analysis of isoflavones. Isoflavone extracts were analyzed using a modular Samples and extraction. Fresh faba beans ( Vicia faba ), peas ( Pisum HPLC system (1100 Series; Agilent Technology, Palo Alto, CA, USA) equipped with a 250×2.0 mm, 4 μm particle diameter, sativum ), ordinary beans ( Phaseolus vulgare ) and soybean ( Glycine ® max ) seeds were purchased at a local market, while dried red clover Jupiter C18 reversed-phase column (Phenomenex, Torrance, CA, (Trifolium pretense L.) was obtained at a herbalist’s shop. Standards USA) that was maintained at 37˚C in a thermostatic oven. of genistin, glycitin, daidzin, biochanin A and formonotenin were Separations were carried out at a 0.2 ml/min constant flow rate, purchased from Sigma-Aldrich (St. Louis, MO, USA). Genistein, applying a 5-65% linear gradient of eluent B (acetonitrile/0.1% glycitein and daidzein aglycones were obtained by acid hydrolysis trifluoroacetic acid) in 60 min, following 5 min of isocratic elution (using 3.4 N HCl at 50˚C for 200 min) of the corresponding β- at 5% B. Eluent A was 0.1% trifluoroacetic acid in HPLC-grade glucosides, according to Chiang et al. (14), with slight water. A diode array detector was used to record UV-Vis spectra modifications. every 2 s in the 190-650 nm range. The HPLC separations were A protocol for the extraction of phenolic compounds was adapted monitored recording at the following wavelengths: λ=254, 280, 320 from Rostagno et al. (15). Briefly, 0.5 g of the dry sample was and 360 nm; and elaborated with the HPLC ChemStation software suspended in 5 ml of 50% ethanol (v/v) and ultrasonicated for 20 vers. A.07.01 (Agilent Technology) furnished with the min at 60˚C. The suspension was centrifuged for 10 min at ~2500×g chromatographer. and the supernatant was recovered. The pellet was re-extracted for 12 hours with an additional 5 ml of solvent and centrifuged. The Cell culture. Three NSCLC cell lines with different EGFR mutation two supernatants were combined and stored in a freezer at −20˚C status and sensitivity to EGFR TKIs were used: HCC827 (del E746- for subsequent analysis (16, 17). A750), NCI-H1975 (T790M), and A549 (wild-type). Cells were obtained from the American Type Culture Collection (ATCC; Determination of total phenolic compounds. The total polyphenol Manassas, VA, USA) and maintained in ATCC-specified growth content in the extracts was determined using the Folin-Ciocolteau medium. method (18). Fifty microliters of dried bean, pea, red clover and soya extracts were combined to 3 ml with distilled water, 250 μl of EGFR inhibitors . Erlotinib and gefitinib were purchased from Folin and 750 μl of 7% (w/v) Na 2CO 3. After 8 min, 950 ml of Genentech (South San Francisco, CA, USA); AstraZeneca (London, distilled water was added. Samples were left for 2 h at room UK) commercial suppliers, respectively. Afatinib was provided by temperature in the dark and the absorbance at 765 nm was then Boehringher Ingelheim (Ingelheim Am Rhein, Rheinland-Pfalz, determined spectrophotometrically microplate reader, (Cary 50 Germany), AZD9291 by AstraZeneca. Stock solutions of TKIs were MPR; Varian, LAB X, LA Jolla, CA, USA). Total polyphenol prepared in dimethyl sulfoxide at a concentration of 10 mmol/l and content was expressed as gallic acid equivalents (GAE, in mg) per maintained at −20˚C. Drugs were diluted to 1 mmol/l in 50% gram of dry material. ethanol for a working solution.

Determination of total flavonoid compounds. Total flavonoid Measure of cellular viability by 3-(4,5-dimethylthiazol-2-yl)-2,5- content was determined using the aluminium chloride colorimetric diphenyltetrazolium bromide (MTT) assay. The three NSCLC cell assay (19). Briefly, 50 μl of extracts or standard solution of catechin lines were seeded at a density of 3,000 cells/well in 96-well plates (6.25, 12.5, 25, 50, 100 μg/ml) in 80% ethanol was added to 10 μl for 24 h. After 24 h, the cells were incubated with different of 10% (w/v) aluminium chloride solution, followed by the addition concentrations of inhibitors (2.5 nM to 15 μM based on cell line of 150 μl of 95% ethanol. Ten microliters of 1 M sodium acetate sensitivity). The effects of each drug alone and the combination of was added to each test sample in a 96-well plate. The absorbance drug and extracts for 48 h were assayed. Cell survival was was measured at 510 nm with a microplate reader (Biotek, San Jose, determined by a standard MTT assay by the addition of 20 μl of

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MTT (Promega CellTiter 96 ® AQueous One Solution Cell; Table I. Total polyphenol and flavonoid content, and antioxidant activity Promega, Madison, WI, USA) for 2 h. The color intensity was of the extracts from peas, fava beans, beans, soybean seeds and red measured by microplate reader (Cary 50 MPR; Varian) at 412 nm. clover. Wells containing cells without any treatment were used as positive controls and the OD value was used to represent 100% cellular Dried sample Total phenols Total flavonoids Inhibition viability. (mg GAE/g) (mg CAE/g) (%)* The half-maximal inhibitory concentration (IC 50 ) values were determined by ED50plus v1.0 online software, (Tlalpan, Mexico Soybean 26.40 17.60 60 Fava bean 37.03 28.52 85 DF, Mexico). Results represent the median of three separate Pea 1.50 0.04 30 experiments each conducted in triplicate. Bean 1.64 0.13 60 Red clover 130.00 71.00 88 Crystal violet assay. Cells were seeded at 3,000 per well in 96-well plates. Twenty-four hours after seeding, cells were exposed to drugs GAE: Gallic acid equivalent; CAE: catechin acid equivalent. *By free with/without extracts as described above. Forty-eight hours after radical-scavenging activity assay. treatment, cells were fixed with formaldehyde and stained with 0.01% crystal violet for 15 min at room temperature. Stained cells were washed with phosphate-buffered saline. Viability was determined by measuring the absorbance at 595 nm (20).

Statistical analysis. ANOVA tests were used for statistical red clover, while total flavonoids ranged from 0.4 mg/g CAE evaluation of results in three independent replications. Values of p for peas to 71 mg/g CAE for red clover. lower than 0.05 were considered significant. The radical-scavenging properties reported as % DPPH • inhibition ranged from 30 to 88%, the highest being that for Results and Discussion red clover extract. Thus, total phenolics, total flavonoids and Isoflavone extracts. Isoflavones, a subclass of the flavonoids, antioxidant potential determined as radical-scavenging are non-nutrient plant compounds, which occur predominantly property were roughly correlated. as β- glucosides (genistin, daidzin, glycitin), or as acetyl- β- glucosides and malonyl- β- glucosides, and are therefore polar, HPLC analysis of isoflavone extracts . The expected water-soluble compounds (21, 22). Precursors of daidzein and isoflavones both in the glycoside and aglycone forms were genistein, and especially the 4’-methyl ethers formononetin and detected in the HPLC chromatogram of the ethanolic extracts biochanin A, respectively, are mainly found in red clover (23). of soybean flour (Figure 1A). Isoflavone components were Like soy, red clover contains the isoflavones genistein, assigned on the basis of the retention time (t r) and UV daidzein, biochanin A, and formononetin; however, soy spectral properties, also in comparison to those of the contains higher amounts of genistein and daidzein, while the standards. Acetyl and malonyl derivatives were identified dominant isoflavones in red clover are biochanin A and through the elution order, by comparison with literature data formononetin (24-27). (32). Isoflavones were also monitored in the extracts from Solvent extraction is the most frequently used technique other legumes, including faba beans, peas and ordinary beans. to obtain crude extracts from matrices of vegetable origin. Considering its well-established pseudo-estrogenic properties, The most common solvents employed are water, ethanol, red clover (Trifolium pratense) was monitored for the acetone and acetic acid, used pure or in a mixture (16). It is occurrence of isoflavones. The HPLC chromatogram of the known that the amount of extractable substances in the ethanolic extracts from dried red clover is shown in Figure course of an extraction with solvents depends both on the 1B. Along with minor peaks corresponding to the isoflavone peculiarities of the raw vegetable material and the solvent components detected in soy, red clover exhibited dominant used (28). Clearly, for each specific plant matrix, there is a peaks at longer t r. By comparison with literature data (33) and need to test and select the solvent and the most suitable on the basis of the UV-Vis spectra, these components were conditions for maximizing the extraction yield and identified as formonotenin (t r 49.9 min; λmax 260, 300 nm) antioxidant activity of the extracts obtained (29-31). For and biochanin A (t r 60.1 min; λmax 248 nm). Analogously to soybean, extraction with acetone/acetic acid was more soy, red clover isoflavones can occur also as acetyl- and efficient, while for red clover, extraction with ethanol malonyl-glucoside derivatives (34). These components are provided the best yield for the extract. most likely the minor peaks with t r in the 35-46 range, Total polyphenols, flavonoid content and antioxidant showing UV spectra characteristic of formonotenin and activities of the extracts from peas, fava beans, beans, biochanin A. A number of additional metabolites structurally soybean seeds and red clover were determined as reported in related to isoflavones have been described (33). However, a Table I. The content of phenolic compounds was found to definitive characterization of these species would require the range from 1.5 mg/g GAE for beans to 130 mg/g GAE for application of complementary analytical techniques. On the

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Figure 1. Reversed-phase high-performance liquid chromatography chromatograms of isoflavones from soy (A) and red clover (B) acquired at λ=254 nm. The main isoflavone components were assigned by spectrophotometric analysis using a diode array detector and comparison with individual standards.

other hand, the HPLC peaks at t r 20-35 min were members AZD9291 was determined by the MTT assay. Both erlotinib, of different metabolite classes and included flavonols such as gefitinib, afatinib and AZD9291 demonstrated growth quercetin glucoside and quercetin rutinoside (rutin) showing inhibition of all three cell lines, but the sensitivity of the cell characteristic absorption bands with λmax =355-360 nm. lines varied markedly. HCC827 cells with mutation in the EGFR gene, (del E746-A750) were highly sensitive to Induction of growth inhibition by erlotinib, gefitinib, afatinib gefitinib and erlotinib at low nanomolar concentrations. A549 and AZD9291. The viability of A549, HCC827 and H1975 cells with wild-type EGFR were sensitive only at higher cell lines treated with gefitinib, erlotinib, afatinib and concentrations of EGFR TKIs (>9 μM), while H1975 cells

5830 Ambrosio et al : Effect of Isoflavones Extracts on NSCLC Cell Lines

were sensitive only at maximum concentrations (>20 μM). Table II. Half-maximal inhibitory concentration (IC 50 ) values for Likewise for the irreversible inhibitors afatinib and AZD9291, tyrosine kinase inhibitors against three cell lines. we observed that HCC827 cells were the most responsive of Median IC 50 ±SD the three cell lines, and H1975 was more responsive to AZD9291 than to afatinib (IC 50 =4.2 μM vs . 12 μM, Cell line Gefitinib Erlotinib Afatinib AZD9291 respectively). After 48 h of treatment, growth of cells was significantly inhibited in a dose-dependent manner and for HCC827 230.0±1.9 nM 70.0±2.2 nM 132.0±0.4 nM 512.0±1.5 nM A549 18.6±1.4 μM 9.2±0.1 μM 11.0±0.4 μM 8.4±0.2 μM each inhibitor, an IC value was calculated from the dose– 50 H1975 48.5±2.8 μM 24.8±1.1 μM 12.0±0.2 μM 4.2±0.7 μM response curves of the NSCLC cell lines. In Table II, IC 50 values for TKI against the three cell lines are reported. SD: Standard deviation.

Soy and red clover isoflavones alone did not exhibit significant growth inhibition. Neither soy nor red clover Effects of erlotinib alone and in combination with extracts extracts had any significant inhibitory effect against all three on NSCLC cell lines. In accordance with the results obtained cell lines, although a trend indicating a reduction of vitality with gefitinib, HCC827 cells were highly sensitive to was found at increasing doses ( p> 0.05). An indicative, albeit erlotinib (IC 50 =70 nM), whereas A549 cells were on average weak, inhibitory effect was observed for HCC827 cells at the sensitive (IC 50 =9.2 μM) and H1975 cells were scarcely highest concentration, not significant in any case ( p> 0.05 responsive (IC 50 =24.8 μM). Due to the presence of the data not shown). T790M mutation, which confers resistance to treatment with TKI inhibitors, H1975 required a higher concentration of Effects of gefitinib alone and in combination with extracts inhibitor compared to the HCC827 and A549 cell lines. on A549, HCC827 and H1975 cell proliferation. To evaluate We observed a reduction of the IC 50 values for all three the effect of GEFITINIB alone and in combination with cell lines when erlotinib was used in combined treatments extracts on the cell viability of A549, HCC827 and H1975 with isoflavones of soy or red clover. Table III reports all the cells in vitro , crystal violet assays were performed for 48 h. IC 50 values. Concentrations above 200 μg/ml of isoflavones, resulted in a marked reduction in cell viability when used in Effects of afatinib alone or in combination with extracts on combination with gefitinib. cellular viability. Dose–response curves for afatinib and It was found that the inhibitory rates with gefitinib alone, extracts were generated for the sensitive EGFR mutant and and in combination with extracts, were higher compared with wild-type NSCLC cell lines. the control group ( p< 0.01). There was no significance The IC 50 values for afatinib combined with soy or red clover difference in vitality between cells treated with the extracts extracts were calculated. HCC827 cells were more responsive alone and control groups ( p> 0.05). However, the inhibitory to afatinib compared with A549 cell line, while H1975 was the rate in the group treated with gefitinib in combination with soy least responsive. For all the cell lines investigated, we observed or red clover extract was higher compared with the agent an enhanced inhibitory effect when TKIs were used in alone ( p< 0.05; Table III). The induction of growth inhibition combination with soy or red clover extracts (Table IV). by gefitinib in combination with red clover extract was higher than that observed with soy extract, although not significantly. Inhibition by AZD9291 irreversible TKI alone and in the The IC 50 value (230 nM) calculated for gefitinib alone presence of isoflavones. Treatment with AZD9291, a third- was reduced (135 nM) in the presence of soy extract and in generation irreversible EGFR TKI, as single agent for 48 h combination with red clover extract (100 nM) for the resulted in a dose-dependent inhibition of growth for HCC827 cell line. HCC827, A549 and H1975 cell lines. The IC 50 values for The combination of isoflavone extracts and gefitinib AZD9291 alone and in combination with soy or red clover showed the highest levels of cytotoxicity, with inhibitory extracts were compared for the three cell lines (Table IV). concentration IC 50 values at 13.47 μM and 12.9 μM for the Soy and red clover extracts markedly synergized AZD9291- A549 cell line, respectively, with soy and red clover extracts, induced cell death, both in HCC827 and H1975, as well as in corresponding order, as compared with drug alone: IC 50 in NSCLC cells with wild-type EGFR (A549). value of 18.6 μM. Ultimately, even for the H1975 cell line carrying the Conclusion mutation which confers drug resistance, the IC 50 value calculated for the drug alone 48.5 μM was reduced to 42 μM Soy and red clover extracts enriched in isoflavones enhanced and 31 μM when the drug was given in combination with cell growth inhibition induced by erlotinib, gefitinib, soy and red clover extract, respectively. afatinib, and AZD9291. Most likely related to its high

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Table III. Half-maximal inhibitory concentration (IC 50 ) values for gefitinib and erlotinib, alone and with soy or red clover extract, against three cell lines.

Median IC 50 ±SD

Gefitinib Erlotinib

Cell line Alone + Soy extract + Red clover extract Alone + Soy extract + Red clover extract

HCC827 230.0±1.9 nM 135.0±6.8 nM 100.0±5.1 nM 70.0±2.2 nM 38.0±3.4 nM 36.0±2.3 nM A549 18.6±1.4 μM 13.5±0.7 μM 12.9±0.2 μM 9.2±0.1 μM 5.8±0.4 μM 5.3±0.4 μM H1975 48.5±2.8 μM 42.0±2.0 μM 31.0±2.0 μM 24.8±1.1 μM 21.7±0.3 μM 20.2±0.9 μM

SD: Standard deviation.

Table IV. Half-maximal inhibitory concentration (IC 50 ) values for afatinib and AZD9291, alone and combined with soy or red clover extract, against three cell lines.

Median IC 50 ±SD

Afatinib AZD9291

Cell line Alone + Soy extract + Red clover extract Alone + Soy extract + Red clover extract

HCC827 132.0±0.4 nM 87.0±0.8 nM 70.0±2.1 nM 512.0±1.5 nM 445.5±8.7 nM 400.0±3.7 nM A549 11.0±0.4 μM 9.6±0.4 μM 6.9±0.6 μM 8.4± 0.2 μM 7.1±0.8 μM 5.2±0.3 μM H1975 12.0±0.2 μM 10.6±0.7 μM 9.8±0.1 μM 4.2±0.7 μM 3.9±0.1 μM 3.5±0.5 μM

SD: Standard deviation.

isoflavone content, red clover extract was the most effective. of the Italian Association of Thoracic Oncology. Clin Lung Soy and red clover isoflavones in combination with TKI Cancer 15 : 173-181, 2014. could represent a potential treatment to contribute to 5 Pao W and Chmielecki J: Rational, biologically based treatment arresting tumor growth or to reducing the doses of the of EGFR -mutant non-small-cell lung cancer. Nat Rev Cancer 10 : chemotherapeutics in the treatment of NSCLC. 760-774, 2010. 6 Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto As far as we are aware, this is the first report on the RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska inhibitory effect of isoflavone extracts and AZD9291 on FG, Louis DN, Christiani DC, Settleman J and Haber DA: NSCLC cell growth. Activating mutations in the epidermal growth factor receptor Further studies are required to elucidate the mechanisms underlying responsiveness of non-small-cell lung cancer to and to confirm the anticancer synergistic activity of TKI- gefitinib. N Engl J Med 350(21) : 2129-2139, 2004. isoflavones in vivo . 7 Maione P, Sacco PC, Sgambato A, Casaluce F, Rossi A and Gridelli C: Overcoming resistance to targeted therapies in References NSCLC: current approaches and clinical application. Ther Adv Med Oncol 7: 263-273, 2015. 1 Hynes NE and Lane HA: ERBB receptors and cancer: the 8 Cross DA, Ashton SE, Ghiorghiu S, Eberlein C, Nebhan CA, complexity of targeted inhibitors. Nature Rev Cancer 5: 341- Spitzler PJ, Orme JP, Finlay MR, Ward RA, Mellor MJ, Hughes 354, 2005. G, Rahi A, Jacobs VN, Red Brewer M, Ichihara E, Sun J, Jin H, 2 Gridelli C, Rossi A, Carbone DP, Guarize J, Karachaliou N, Mok Ballard P, Al-Kadhimi K, Rowlinson R, Klinowska T, Richmond T, Petrella F, Spaggiari L and Rosell R: Non small cell lung GH, Cantarini M, Kim DW, Ranson MR and Pao W: AZD9291, cancer. Nature Rev Dis Primers 1: 1-16, 2015. an irreversible EGFR TKI, overcomes T790M-mediated 3 Giaccone G: Epidermal growth factor receptor inhibitors in the resistance to EGFR inhibitors in lung cancer. Cancer Discov treatment of non-small-cell lung cancer. J Clin Oncol 23 : 3235- 4(9) : 1046-1061, 2014. 3242, 2005. 9 Jänne PA, Yang J C, Kim DW, Planchard D, Ohe Y, Ramalingam 4 Gridelli C, de Marinis F, Cappuzzo F, Di Maio M, Hirsch F, SS, Ahn MJ, Kim SW, Su WC, Horn L, Haggstrom D, Felip E, Mok T, Morgillo F, Rosell R, Spigel D, Yang J and Ciardiello F: Kim JH, Frewer P, Cantarini M, Brown KH, Dickinson PA, Treatment of advanced non small cell lung cancer with Ghiorghiu S and Ranson M: AZD9291 in EGFR inhibitor- epidermal growth factor receptor ( EGFR ) mutation or ALK gene resistant non small-cell lung cancer. N Engl J Med 372(18) : rearrangement: results of an International Expert Panel Meeting 1689-1699, 2015.

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